Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human disease. With the continued emergence of multi-drug resistant isolates, the identification of novel targets is crucial in our fight against a return to the pre-antibiotic era. The success of S. aureus as a pathogen is largely due to its vast array of virulence determinants, or more specifically, the complex network of regulatory elements that modulate these and other genetic components. From the perspective of pathogenesis, this network of control presumably exists to fine-tune the requirements of the organism within the host environment. We have recently identified a novel component of this regulatory network: an alternative sigma factor (CS) which is required not only for S. aureus adaptation and survival, but is also vital for its virulence. Using a murine model of septic arthritis we found that mice infected with a sigS mutant displayed significantly decreased weight-loss, mortality, severity of infection, systemic dissemination and mounted immune responses. Thus our specific hypothesis is that CS represents a major missing component of the S. aureus virulence regulatory network. We contend that the thorough analysis of its function and control proposed in this application will ultimately enable us to better understand the pathogenesis of this organism, and may aid in the rational development of novel therapeutic treatments. To this end we propose to: 1. Analyze the mechanisms required for sigS induction in S. aureus. We will use promoter mapping, DNA- protein pulldown assays, and high-throughput reporter gene fusion technologies coupled with growth manipulation to determine the exact mechanisms responsible for the induction of CS activity in S. aureus. 2. Define the function of CS in the S. aureus stress and virulence responses. We will use microarray analysis to determine exactly which genes are controlled by CS; and phenotypic microarrays to probe the metabolic and physiological contributions made by CS to S. aureus biology. 3. Investigate the specific pathways that control CS activity in S. aureus. ECF-sigma factors are commonly regulated in a post- translational manner via a complex cascade of protein interaction, mediated by inhibition and proteolysis. We will probe S. aureus specific components of the CS regulatory network using gene-transcription technologies, specific protein analysis, yeast-2-hybrid studies and mutagenic screens. With infections caused by S. aureus on the rise globally it is vital that every effort is exerted to understand the mechanisms involved in its pathogenesis. The investigations proposed here will provide us with a unique and specific insight into the molecular mechanisms of S. aureus disease, by mapping the function and control of a major new virulence regulator. The knowledge derived may also aid in the rational design of novel anti-staphylococcal therapies. Finally, given the independent evolutionary origins of CS in the staphylococci, our investigations will likely deliver new information regarding prokaryotic regulatory networks. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human infection. With the continued emergence of multi-drug resistant isolates of S. aureus (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal seeks to investigate and understand how a novel regulator of virulence, CS, contributes to disease causation in S. aureus.